(1) Field of the Invention
This invention is directed generally to improvements to the analysis of chemical compounds using mass spectroscopy methods. More specifically, the invention relates to ferrocenyl boronate derivatization of chemical compounds to be analyzed and to their subsequent analysis using electrospray tandem mass spectrometry methods.
(2) Description of the Related Art
Mass spectroscopy is a well-known tool used for analyzing chemical compounds. Tandem mass spectroscopy, using electrospray ionization, has been used with increasing frequency for the analysis of biological samples. Persons skilled in analytical chemistry methods are familiar with the operation of electrospray tandem mass spectrometry instruments.
Currently a need exists for improved methodologies enabling one to chemically or mechanically manipulate large numbers of compounds in order to extract subtle differences in structure. A number of complex carbohydrates and esters are biologically significant molecules in that they contain a high degree of information per molecular unit and constitute a prevalent form of protein post-translational modification. (R. A. Dwek Biochemical Society Transactions 1995; 23: 1.) It is desirable not only to obtain amino acid sequences of a number of glycoproteins, but also to investigate the substance and microheterogeneity of certain portions of these molecules. (M. E. R. O""Brien, B. E. Souberbielle, M. E. Cowan, C. A. Allen, D. M. Luesley, J. J. Mould, G. R. P. Blackledge, G. R. B. Skinner Cancer Letters 1991; 58: 247.) Informational properties of chemical moieties are dependent on their high degree of conformational and isomeric diversity resulting from subtle changes in unit assembly. (R. A. Laine, Glycobiology 1994; 4: 749.) Changes in compound architecture often reflect major biological implications, including disease. (S. I. Hakormori Cancer Research 1985; 45: 2405; A. Eiras-Segal, M. V. Croce, Allegol. Et. Immunopathol. 1998; 25: 176; I. Brockhausen, Biochemical Society Transactions 1997; 25: 871; C. M. Martersteck, N. L. Kedersha, D. Drapp, T. Tsui, K. J. Colley, Glycobiology 1996; 6: 289; D. Naor, R. V. Sionov, D. Ish-Shalom, Cancer Research 1997; 71; 241; K. O. Lloyd, Cancer Biology 1991; 2: 421; S. Leppa, J. Heino; M. Jalkanen, Cell Growth and Differentiation 1995; 6: 853.) As a result, the number of potential biologically significant structures is significant. (B. Fernandes, U. Sagman, M. Auger, M. Demnetrio, J. W. Dennis, Cancer Research 1991; 51: 718.)
Structural elucidation of carbohydrates has been accomplished using a combination of techniques, usually nuclear magnetic resonance (NMR) and mass spectrometry (MS). These techniques have proven labor intensive. (J. F. Kennedy, G. Pagliuca, Oligosaccharides: Second Edition ed.; J. F. Kennedy, G. Pagliuca, Ed.; IRL Press at Oxford University Press: Oxford, New York, Tokyo, 1994, pp. 43-68).
Tandem MS, particularly clectrospray quadruple ion-trap MS (ES-MS) is well suited for examining complex mixtures, particularly when coupled with a front-end separation system such as liquid chromatography (LC-MS). (N. Kawasaki,; M. Ohta; S. Hyuga; O. Hashimoto; T. Hayakawa. Analytical Biochemistry 1999, 269, 297-303; M. Kohler; J. Leary. Analytical Chemistry 1995, 67, 3501-3508; H. Kwon, J. Kim. Journal of Liquid Chromatography 1995, 18, 1437-1449). ES-MS offers the possibility of working efficiently and with lower amounts of sample without the possibility of thermal losses, which can often be a requirement for the study of biological structure and function relationships. (N. H. Packer, M. J. Harrison, Electrophoresis 1998; 19: 1872). Great potential exists for ES-MS methods eliciting fine structural and relevant details in larger chemical units. (N. Viseux, E. de Hoffmann, B. Domon, Anal. Chem. 1998; 70: 4951.)
Peptide sequencing by MS methods has rapidly developed into a mature science. (D. F. Hunt, J. E. Alexander, L. Ashley, P. A. McCormack, H. M. Martino, J. Shabanowitz, N. Sherman, M. A. Mosely, J. W. Jorgenson, K. B. Tomer, Mass Spectromebric Methods for Protein and Peptide Sequence Analysis; Academic Pres, Inc.: San Diego, Calif., 1991.) On the other hand, because of the large differences in fragmentation energies found in carbohydrates, the application of these types of methods in the analysis of carbohydrates has been limited. Also, due to the poor ionization in ES-MS of information-rich sugars released from their parent molecule by application of enzymatic deglycosylation, this severely affects sensitivity and consequently hinders detection.
Many analytical methods for analysis of compounds involve some form of derivatization to enhance sensitivity or fragment information in the mass spectrometer. The analysis of certain compounds has relied on derivatization chemistry to enhance the sensitivity of these important biological compounds as applied to their specific techniques. (R. A. Dwek, Biochemical Society Transactions 1995, 23 1-25; C. M. Starr, R. I. Masada, C. Hague, E. Skop, J. C. Klock, J. Chromatogr A 1996, 720, 295-321; M. F. Chaplin, Monosaccharides; Second Edition; M. R. Chaplin, Ed. IRL Press at Oxford University Press: Oxford, New York, Tokyo, 1994, pp 1-40.) Chemical derivatization has been employed to overcome the ionization quandry. (D. J. Harvey, J. Am. Soc. Mass Spectrom. 2000, 11, 900-915; J. J. Pitt, J. Gorman, Analytical Biochemistry 1997, 248, 63-75; D. Williams, T. D. Lee, N. Dinh, M. K. Young, Rapid Commun. Mass Spectrom 2000, 14, 1530-1537; S. Susuki, K. Kakehi, S. Honda, Anal Chem 1996, 68, 2073-2083.)
Almost all methods of MS analysis of compounds rely heavily on some form of derivatization not only to increase sensitivity, but also to augment volatility and the proclivity to form useful fragment ions. Derivatization must not only increase the propensity of the analyte to ionize, but also provide the desired quality of fragment information during successive rounds of ES-MS. Desirable fragments should provide both selective loss of chemical units and, within these fragments, cross pyranose ring fissions should reflect subtle stereochemical differences between individual molecular units. (H. Desaire, J. A. Leary, Anal. Chem. 1999, 71, 1997-2002; Z. Zhou, S. Ogden, J. A. Leary J. Org. Chem. 1990, 55, 5444-5446; S. P. Gaucher, J. A. Leary Anatylical Chemistry 1998, 70, 3009-3014.) Cross pyranose ring fragmentation releases neutral fragments. The parent ion then characterizes the linkage position between molecular units. As a result, profiling, sequence identity and linkage position information may be explored. Normally, the larger molecules or compounds, particularly those that contain nitrogen in the form of N-acetylated aminohexose residues, can be electrosprayed with or without permethylation. However, the MS2 fragmentation information is often not particularly informative due to simple water loss. This technique works even less reliably with smaller molecules found during enzymatic digestion protocols. As the molecule becomes smaller and contains fewer chargeable atoms (usually nitrogen) and a diminished propensity for cationization, the efficiency of ES-MS analysis declines. Considerable attention has been paid to various methods of mono and oligosaccharide complexation with a variety of metal ions. (H. Desaire,; J. A. Leary, Anal. Chem. 1999; 71: 1997; S. Konig, J. A. Leary, Journal of the American Society for Mass Spectrometry 1998; 9: 1125; G. Smith, J. A. Leary Journal of the American Society for Mass Spectrometry 1996; 7: 953; Z. Zhou, S. Ogden, J. A. Leary, J. Org. Chem. 1990; 44: 5444; M. Kohler, J. A. Leary, Analytical Chemistry 1995; 67: 3501; G. F. Hofineister, Z. Zhou, J. A. Leary, J. Am. Chem. Soc. 1991; 113: 5964.)
Furthermore, in certain cancer studies, the catechol estrogens 2-hydroxyestradiol (2-OHE) and 4-hydroxyestradiol (4-OHE) are implicated as so-called good and bad estrogens in the biogenesis of malignant cells (S. H. Safe, Interactions Between Hormones and Chemicals in Breast Cancer, Ann. Rev. Pharmacol. Toxicol., 1998. 38: p. 121-158; E. L. Cavalieri, et al., Molecular Origin of Cancer: Catechol Estrogen-3,4-Quinones as Endogenous Tumor Initiatiors Proc. Natl. Acad. Sci. USA, 1997. 94: 10937-10942; D. E. Stack, Molecular Characteristics of Cathechol Estrogen Quinones in Reactions with Deoxyrbonucleosides Chem. Res. Toxicol., 1996 9: 851-859; L. Shen et al., Bioreductive Activation of Catechol Estrogen-ortho-quinones: Aromatization of the B ring in 4-Hydroxyequilenin markedly Alters Quinoid Formation and Reactivity. Carcinogenesis, 1997. 18(5): 1093-1101.) It has been demonstrated that these compounds can be formed by cytochrome P450 hydroxylation of estradiol (E. L. Cavalieri, et al.). Modulation of these products is controlled through the action of catechol-O-methyl transferases, which methylate the newly introduced phenolic hydroxyl group. The so-called good estrogen (2-OHE) is thought to perform important positive roles within the cell such as the suppression of osteoporosis and atherosclerosis because of its ability to inhibit leucotriene synthesis, (J. Alanko, et al., Catechol Estrogens as Inhibitors of Leucotriene Synthesis. Biochemical Pharmacology, 1998. 55: 101-104.) a potent stimulator of bone readsorption. On the other hand, 4-OHE is prone to easy oxidation aid if it avoids methylation, glucuronidiation, sulfation or other neutralizing processes it may become oxidized to the semiquinone. The semiquinone is redox reactive and has been shown to depurinate DNA and produce significantly increased numbers of mammary cancers in animal models (L. Shen et al.; N. T. Telang, Estradiol Metabolism: A Endocrine Biomarker for Modulation of Human Mammary Carcinogenesis. Environmental Health Perspectives, 1997. 105 (3): 559-564.) An analogous mechanism has been postulated for the development of prostate cancer (J. F. Dorgan, et al., Relationships of Androgens and Estrogens to Prostate Cancer Risk Results from a Prospective Study in Finland. Cancer Epidemiology, Biomarkers and Prevention, 19987: 1069-1074.)
To better understand the links between these two bio-effector molecules in the context of both breast and prostate cancer, sensitive methods of analysis for these compounds are essential. To date the most common form of chemical analysis for catechol estrogens has been gas chromatography using mass spectrometric detection (GC-MS) or radiometric (tritiur release). (Jordan, S. W., I. S. Krull, and S. B. J. Smith, The Trace Analysis for Catechol Derivatives via Boronate Ester Formation and GC-Microwave Induced Plasma Emission Spectroscopic Detection (GC-MIP). Analytical Letters, 1982. 15(A14): p. 1131-1148; Sepkovic, D. W., et al, Catechol Estrogen Production in Rat Microsomes after Treatment with Indole-3-Carbinol, Ascorbigen, or xcex2-Napthoflavone: A Comparison of Stable Isotope Dilution Gas Chromatography-Mass Spectrometry and Radiometric Methods. Steroids, 1994. 59: p. 318-323.). These methods, although sensitive, all possess certain drawbacks. Electrospray mass spectrometry (ES-MS) has proven to be a robust and sensitive method for the detection and quantitation of both large and small molecules. (Cole, R. B., ed. Electrospray Ionization Mass Spectrometry. Fundamentals, Instrumentation, and Applications. 1 ed. 1997, John Wiley: New York, Chichester, Weinheim, Brisbane, Singapore, Toronto. 577.) However, most analyses involve aqueous organic liquid mixtures containing analytes that have easily ionizable functionalities. The analysis of neutral lipophilic compounds such as 2- and 4-OHE requires the use of a suitably non-polar solvent and a methodology for creating a charged species.
The present invention provides a sensitive and semi-quantitative method for analyzing both hydroxyestradiol isomers. The methodology utilizes ferrocenyl boronic acid derivatives (Brooks, C. J. W. and W. J. Cole, Analytical Separation and Characterization of 1,2 and 1,3 diols as their Cyclic Ferrocene Boronate Derivatives. Journal of Chromatography, 1986. 362: p. 113-116; Brooks, C. J. W. and W. J. Cole, Cyclic Ferroceneboronates as Derivatives for the Gas Chromatographic Separation and Characterisation of Diols and Related Compounds. Journal of Chromatography, 1987. 399: p. 207-221; Van Berkel, G. J., et at, Derivatization for Electrospray Ionizable Mass Spectrometry. 3. Electrochemically Ionizable Derivatives. Anal Chem, 1998. 70: p. 1544-1554; Van Berkel, G. J., et al, Derivatization for Electrospray Ionization Mass Spectrometry. 3. Electrochemically Ionizable Derivatives. Anal Chem., 1998. 70: p. 1544-1554) that undergo single-electron oxidation when sprayed from an ES interface having a large surface area electrode (Van Berkel, G. J. and F. Zhou, Characterization of an Electrospray Ion Source as a Controlled-Current Electrolytic Cell. Anal Chem, 1995. 67: p. 2916-2923; Van Berkel, G. J. and F. Zhou, Electrochemistry Combined On-Line with Electrospray Mass Spectrometry. Anal Chem, 1995. 67: p. 3643-3649; Van Berkel, G. J., S. A. McLuckey, and G. L. Glish, Preforming Ions in a Solution via Charge-Transfer Complexation for Analysis by Electrospray Ionization Mass Spectrometry. Analytical Chemistry, 1991. 63(18): p. 2064-2068.) Fragmentation spectra of the radical cation molecular ions of the two isomers are sufficiently different that their relative ratios in a mixture can be determined.
In accordance with one aspect of the invention, there is provided a new and improved method for analyzing chemical species using mass spectrometry. One or more species to be analyzed are labeled (derivatized) with ferrocenyl boronate and the labeled compounds are introduced into a mass spectrometer instrument. In preferred embodiments, the mass spectrometer uses electrospray tandem technology.
Another aspect of the invention relates to an inventive spray needle that acts as a controlled current electrochemical cell for an electrospray tandem MS instrument. The needle produced fragment ion spectrum to aid in identifying and analyzing compounds for subtle changes in structure.
A variety of chemical compounds can be analyzed in accordance with the present invention, including N-acetylated hexose carbohydrates (such as 2-N-acetamido-D-glucosamine, 2-N-acetamido mannosamine, 2-N-acetamidogalactosamine and lactofucsylatraose). Alternatively, the chemical compound may be a neutral isomeric low molecular weight carbohydrate such as an aldohexose, 6-dideoxyaldohexose where the aldohexoses and 6-dideoxyaldohexosis are preferably D-glucose, D-mannose, D-galactose, L-fructose or L-rhamnose. The chemical compound may be a disaccharide such as maltose, cellobiose, lactose, D-Glc disaccharide or an O-methyl glycoside. Other examples include catechol estrogens including 2-hydroxyestradiol and 4-hydroxyestradiol.